Ann E. Blechl

A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat.

Enhancing the nutritional value of food crops is a means of improving human nutrition and health. We report here the positional cloning of Gpc-B1, a wheat quantitative trait locus associated with increased grain protein, zinc, and iron content. The ancestral wild wheat allele encodes a NAC transcription factor (NAM-B1) that accelerates senescence and increases nutrient remobilization from leaves to developing grains, whereas modern wheat varieties carry a nonfunctional NAM-B1 allele. Reduction in RNA levels of the multiple NAM homologs by RNA interference delayed senescence by more than 3 weeks and reduced wheat grain protein, zinc, and iron content by more than 30%.

The wheat and barley vernalization gene VRN3 is an orthologue of FT

Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate flowering (vernalization), whereas spring varieties do not have this requirement. In this study, we show that in these species, the vernalization gene VRN3 is linked completely to a gene similar to Arabidopsis FLOWERING LOCUS T (FT). FT induction in the leaves results in a transmissible signal that promotes flowering. Transcript levels of the barley and wheat orthologues, designated as HvFT and TaFT, respectively, are significantly higher in plants homozygous for the dominant Vrn3 alleles (early flowering) than in plants homozygous for the recessive vrn3 alleles (late flowering). In wheat, the dominant Vrn3 allele is associated with the insertion of a retroelement in the TaFT promoter, whereas in barley, mutations in the HvFT first intron differentiate plants with dominant and recessive VRN3 alleles. Winter wheat plants transformed with the TaFT allele carrying the promoter retroelement insertion flowered significantly earlier than nontransgenic plants, supporting the identity between TaFT and VRN-B3. Statistical analyses of flowering times confirmed the presence of significant interactions between vernalization and FT allelic classes in both wheat and barley (P < 0.0001). These interactions were supported further by the observed up-regulation of HvFT transcript levels by vernalization in barley winter plants (P = 0.002). These results confirmed that the wheat and barley FT genes are responsible for natural allelic variation in vernalization requirement, providing additional sources of adaptive diversity to these economically important crops.

Rapid Production of Multiple Independent Lines of Fertile Transgenic Wheat (Triticum aestivum)

Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.

A Kinase-START Gene Confers Temperature-Dependent Resistance to Wheat Stripe Rust

Stripe rust is a devastating fungal disease that afflicts wheat in many regions of the world. New races of Puccinia striiformis, the pathogen responsible for this disease, have overcome most of the known race-specific resistance genes. We report the map-based cloning of the gene Yr36 (WKS1), which confers resistance to a broad spectrum of stripe rust races at relatively high temperatures (25° to 35°C). This gene includes a kinase and a putative START lipid-binding domain. Five independent mutations and transgenic complementation confirmed that both domains are necessary to confer resistance. Yr36 is present in wild wheat but is absent in modern pasta and bread wheat varieties, and therefore it can now be used to improve resistance to stripe rust in a broad set of varieties.

Activity of a maize ubiquitin promoter in transgenic rice.

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat.

Vernalization, the requirement of a long exposure to low temperatures to accelerate flowering, is an essential adaptation of plants to cold winters. The vernalization gene VRN-1 plays an important role in this process in diploid (Triticum monococcum) and polyploid wheat (Triticum aestivum). We have recently shown that the diploid wheat VRN-Am1 gene was similar to the Arabidopsis (Arabidopsis thaliana L. Heynh.) APETALA1 meristem identity gene. We also showed that dominant Vrn-Am1 alleles were the result of loss-of-function mutations in regulatory regions recognized by a VRN-1 repressor, likely VRN-2. This model predicts that only the dominant Vrn-1 allele will be transcribed in lines carrying both recessive and dominant alleles. Here, we confirm this prediction in young isogenic lines of hexaploid wheat carrying different dominant Vrn-A1, Vrn-B1, and Vrn-D1 alleles, and also in heterozygous VRN-1 diploid wheat plants. However, a few weeks later, transcripts from the recessive alleles were also detected in both the polyploid and heterozygous diploid spring plants. Transcription of the recessive alleles was preceded by a reduction of the transcript levels of VRN-2. These results suggest that the dominant Vrn-1 allele or a gene regulated by VRN-1 down-regulates the VRN-2 repressor facilitating the transcription of the recessive alleles in unvernalized plants. We also show here that the level of VRN-1 transcripts in early developmental stages is critical for flowering initiation. A reduction of VRN-1 transcript levels by RNA interference delayed apex transition to the reproductive stage, increased the number of leaves, and delayed heading time by 2 to 3 weeks. We hypothesize that the coordinated transcription of dominant and recessive alleles may contribute to an earlier attainment of the VRN-1 transcript level threshold required to trigger flowering initiation in polyploid wheat.

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene

Abstract.Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

The Expression of a Bean PGIP in Transgenic Wheat Confers Increased Resistance to the Fungal Pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plants ability to inhibit the pathogens capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheats defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.

Advancing Crop Transformation in the Era of Genome Editing

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

RNA interference for wheat functional gene analysis

RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this approach is in its early stages, but has great potential since multiple homoeologous copies can be simultaneously silenced with a single RNAi construct. In this article, we discuss the utilization of RNAi in wheat functional gene analysis and its effect on transcript regulation of homoeologous genes. We also review recent examples of RNAi modification of important agronomic and quality traits in wheat and discuss future directions for this technology.